An important objective in the analysis of a sample with a scanning microscope is to determine the quantitative information about the sample. This information can relate, for example, to the spatial distribution of fluorescent molecules, in particular, the number or density of fluorescent emitters.
The light intensity, emitted by fluorescent molecules or other sample structures of interest, is often measured; and this light intensity depends monotonically on the number or density of the molecules of interest. However, in this case only relative quantitative statements are easily possible with such a measurement technique. That is, a determined number or density is given in arbitrary units. Therefore, it may be expedient to carry out a quantitative comparison only with measured data from the same measurement.
In fluorescence correlation microscopy (FCS) fluctuations in the fluorescence intensities are analyzed by means of a correlation analysis. This approach allows an absolutely quantitative analysis, in which a density or concentration of substances of interest is not given in arbitrary units, but in physical quantities. However, precise knowledge of the observed sample volume is required for a reliable interpretation of the data, i.e., precise knowledge of the sample volume that contributes to a signal from a pixel of a recorded sample image.
The sample volume can be computed, if the point spread function, with which the sample analysis is performed, is known. The point spread function (PSF) is also referred to as a spatial response or blurring function.
Even in the case of other interpretation methods, knowledge of the analyzed sample volume is required to determine the absolute quantitative values, which can be done by means of the point spread function.
The point spread function depends on all of the elements in the optical path of the microscope. In principle, it is possible to compute a theoretical PSF, if the optical parameters of the elements in the beam path are known. However, the actual PSF can deviate from the theoretical PSF, for example, due to the production dependent fluctuations in the optical elements; due to the variances in the use of the elements; due to the disregarded variances in the immersion medium, which is used with respect to an immersion medium, which is used in the computation; or due to the variances in the temperature of the optical elements with respect to a temperature that is assumed in the computation.
Therefore, more accurate results can be obtained, if the PSF is determined by experiment. For this purpose, it is possible to analyze, in particular, a reference sample that comprises objects, so-called beads, that are smaller than the resolution of the scanning microscope. The full width at half maximum (FWHM) of the PSF may be regarded as the resolution.
Although the PSF for the objective lens that is used can be determined with a high degree of accuracy by such a reference measurement, said reference measurement does not take into consideration the elements, which are associated with the actual sample, for example, the immersion medium and the temperature dependence of the refractive index thereof. This temperature dependence can be high, especially with oil as the immersion medium. Moreover, any variance in the thickness of a cover glass that is used is not taken into consideration. This can lead to a spherical aberration. Any inclination of the cover glass may lead to astigmatism and coma of the PSF.
The error in a computed PSF or a PSF, determined in a reference measurement, with respect to the actual PSF for the analysis of the actual sample may be large, in particular, in the vertical direction. The vertical direction is supposed to denote the direction of the optical axis that runs from an objective lens to the sample. As a result, the sample volume, analyzed with a detector element, can vary by a factor of 2 or more of a computed volume.
For these reasons it is advantageous if the PSF is not determined from a reference measurement, but rather experimentally on the actual sample.
This idea forms the basis for a method for operating a scanning microscope and for determining point spread functions, with which sample images are recorded with the scanning microscope. In such a method it is provided that a sample is scanned with at least one illuminating light beam; that at least one sample image is recorded with a detector device of the scanning microscope during a scan by the illuminating light beam; and that the point spread function, with which a sample image is recorded by the scanning microscope, is computed from the at least one sample image.
A scanning microscope conforming of this type includes a light source device for emitting at least one illuminating light beam, a scanning device for generating a scanning motion of the at least one illuminating light beam across a sample, a detector device for recording at least one sample image during a scan by the illuminating light beam, and electronic control and evaluation means, which are designed to compute a point spread function, with which a sample image is recorded, from the at least one sample image.
In a method known from the prior art, reference objects, such as beads, which are smaller than the microscope resolution, are added to the sample. The PSF with respect to a sample image can be determined from the detector signals with respect to these reference objects. The drawback with this method is that more work is required to prepare the samples.